Shotgun method for massive mutational interrogation of the entire human genome PROJECT SUMMARY/ABSTRACT Massive mutational interrogation of the entire genome of an organism is a powerful genetic tool; it has fueled forward genetic studies in model organisms for several decades with great success. Since a substantial fraction of human genes is multi-functional, traditional knockout/knockdown approaches invariably perturb all functions of such genes. In contrast, massive mutational interrogation allows separating distinct functions of genes, as is routinely done in model organisms. Revolutionary CRISPR-Cas technology has provided a long- missing tool for introducing mutations into the human genome, enabling the era of human genome-wide mutational forward genetics. Unfortunately, all CRISPR-based genome interrogation approaches have one significant limitation: they are only as comprehensive as the sgRNA libraries that they employ. To date, technical limitations have constrained the sizes of even the largest reported CRISPR sgRNA libraries to about 105 sgRNAs, precluding dense mutational interrogation of the entire human genome. In contrast, ultra-high throughput FACS-based screening methods (including our Fireworks method) provide tremendous screening capacity for fluorescence- based phenotypes. This screening capacity vastly exceeds the sizes of existing sgRNA libraries, enabling a fundamental change in the way the human diploid genome is interrogated. I am demonstrating proof-of-principle of the Shotgun method for in-vivo mutational analysis of the entire human genome. The Shotgun method increases the genome-wide density of CRISPR-based mutational interrogation more than 400-fold over existing methods. First, the genome is densely targeted with billions of different sgRNAs, creating an immense library of mutations. Then, phenotypes of the resulting mutants are interrogated individually using high-speed FACS sorting. The lentiviral sgRNA library I constructed is 4(!) orders of magnitude larger than the largest reported sgRNA libraries; its diversity is constrained by the theoretical limit of the sequence space of random 16-mers within sgRNAs (416 = 4.3x109). I developed a protocol and currently use an efficient screening of this library in vivo to identify sgRNAs that inhibit nonsense- mediated decay (NMD); I demonstrate completion of one FACS screening round of 5.2x109 (5.2 billion) individual randomized sgRNAs in human (diploid) cells in just 4 days. By virtue of its design, the Shotgun method interrogates the entire 3.2 Gb of the human genome, on average, every 48 base pairs. As a result, Shotgun targets all known and unknown genomic elements: exons, introns, promoters, 3?? UTRs, as well as non-coding and intergenic regions, providing the most unbiased and comprehensive tool for mutational analysis of the diploid human genome to date. This project aims to capitalize on the immense mutational coverage of the human genome provided by the Shotgun approach by identifying, analyzing, and validating the resulting hits for their roles in the NMD pathway.